N. gonorrhoeae, the causative agent of gonorrhea, is responsible for -300,000 infections in the U.S. and over 6 million globally. Antibiotics remain the primary treatment for gonorrhea infections, but antibiotic resistance threatens their continued use. Penicillin and tetracycline were once the antibiotics of choice but are no longer effective, and resistance to fluoroquinolones is rapidly increasing. My laboratory focuses on the molecular mechanisms of antibiotic action in the gonococcus, particularly the [unreadable]-lactam antibiotics such as penicillin, and how resistance arises to these drugs, which in N. gonorrhoeae is an unusually complex and multifactorial process that is not completely understood. We have recently uncovered a surprising role in antibiotic influx of the PilQ secretin, one of the proteins in the Type IV pilus complex, which is involved in adhesion and invasion, twitching motility, and DNA transformation. In Specific Aims 1 and 2, we will follow up on this discovery to establish how PilQ and other Type IV pilus proteins promote the entry of antibiotics, and to define the interactions of these proteins with each other in the Type IV pilus complex. In Specific Aim 3, we outline experiments to clone and characterize a recently identified resistance determinant found in high- level penicillin-resistant clinical isolates. This aim will provide new understanding of the mechanisms of chromosomally mediated resistance and novel insight into gonococcal physiology. In Specific Aim 4, we describe a proteomics approach to identify and characterize proteins that bind to penicillin-binding protein 1 (PBP 1), PBP 2, and the lytic transglycosylase MltA, and thus reveal the form and function of the multi-enzyme complexes that synthesize peptidoglycan. Together, these projects utilize genetic, biochemical, biophysical, and proteomics approaches to generate new insights into the mechanisms of antibiotic action in N. gonorrhoeae.